The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. In a separate project, we discovered DNA sequence motifs that are preferentially bound by different homeodomain (HD) transcription factors, which suggests a novel mechanism for how HD-specific transcriptional responses might be generated during embryonic development. In particular, we have tested the hypothesis that HD-preferred sites for the FC identity HD (FCI-HD) protein Slouch (Slou) serve a critical regulatory function in determining the specificity of muscle founder cell (FC) gene expression mediated by Slou at single cell resolution using previously characterized Slou-responsive FC enhancers as an experimental model. In these studies, we mutagenized a single Slou-preferred binding site in each of two founder myoblast enhancers that are active in Slou-expressing FCs, and then assayed the effects of these mutations in vivo using transgenic reporter assays in which an otherwise isogenic wild-type construct with the identical insertion site served as a reference control. These experiments revealed that Slou-preferred binding sequences make a critical contribution to the regulatory specificity exhibited by Slou in the cells in which that HD is expressed, a findng that correlates with the muscle identity function of Slou. In addition, both activating and repressing functions of Slou were found to be mediated by Slou-preferred binding sites in different FC enhancers. Furthermore, Slou-responsive enhancers containing functionally relevant Slou-preferred motifs were associated with genes that act at both upstream regulatory and downstream effector steps of myogenesis, suggesting that this novel mechanism for mediating the specificity of Slou activity is applicable to both proximal and distal nodes of the regulatory network in which Slou participates. Additional evidence for the functional significance of Slou-preferred binding sites derived from experiments in which these sequences were swapped for sites that are capable of binding to multiple HD family members, a manipulation that does not support the normal regulatory functions of Slou. We also demonstrated that not all Slou-preferred sites exert the same effects when substituted in the identical location within a particular enhancer. Thus, the precise nucleotide sequence of a Slou binding site is crucial for the function of this HD TF, a conclusion that is further supported by the high degree of evolutionary conservation of the Slou-preferred binding sites whose functions we validated. To extend our experiments on a limited number of enhancers containing Slou-preferred binding sites, we conducted computational analyses in an attempt to generalize the abovementioned empirical results. To accomplish this goal, we examined whether Slou- and Msh-preferred binding sites are over-represented in putative enhancers associated with genes that are responsive to the corresponding HD TF. These large-scale scans employed modified versions of the PhylCRM and Lever algorithms that were originally developed by our collaborators in Dr. Martha Bulyks laboratory at Harvard Medical School and Brigham and Womens Hospital. The findings of these computational studies revealed that Slou- and Msh-preferred bindings sites are indeed enriched in combination with other classes of regulatory motifs that together comprise candidate enhancers associated with gene sets that are responsive to the corresponding HD TF, but a similar enrichment is not observed in equivalent regions associated with sets of control genes that we determined are not regulated by the same HD proteins. Thus, an independent computational strategy that is complementary to more conventional experimental assays yielded results that support the generality of a previously unrecognized mode of transcriptional regulation by the developmentally important class of HD TFs. In this way, multiple convergent lines of evidence support the existence of a novel molecular mechanism for HD regulatory specificity. Taken together, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and provide strong evidence that the diversity of HD binding preferences can confer specificity to the transcriptional regulation that is mediated by this category of TF.